Acanthamoeba are ubiquitous protists that play an environmental role in regulating microbial diversity; they also occasionally cause infections of the eye (Acanthamoeba keratitis) and brain (granulomatous amoebic encephalitis). These organisms exhibit two distinct phenotypes. The trophozoite form dominates in favorable conditions, in which the Acanthamoeba move through the extension of pseudopodia, engulfing microbes and other particles. During stressful conditions, the Acanthamoeba undergo a process of encystment, in which they build a double cell wall and become relatively inactive. The cyst form can survive years until more favorable conditions arise, at which point they may excyst. For this study, multiple laboratory encystment methods were compared to determine the percent encystment and the different viabilities of laboratory-produced cysts. Furthermore, four different encystment genes were targeted for development of a primer library for reverse-transcription, polymerase chain reaction expression studies. The library was developed using sequences accessed from various databases, including NCBI and EMBL; primers were screened through polymerase chain reaction, and those primers producing positive results were used to further screen cellular RNA that was extracted from encysting cells over various time points during the encystment process, and using various encystment media. Using these methods, target gene involvement in the encystment process was compared between species and encystment methods. These studies lay the foundation for quantitative gene expression analysis, and provide the basis for comparison of various encystment methods.

To establish the gene expression profile and cascade subsequently on avian sex-determining candidate genes, seven avian sex-determining candidate genes including DMRT1, FET1, FOXL2, LHX9, HINT1, SMC2L1 and SOX9 were analyzed at early embryogenesis. Quantitative reverse transcription PCR (Quantitative RT-PCR) technology was used to establish the gene expression profiles among these genes at four, five, six and seven days of embryos. The results of quantitative RT-PCR reveal that the DMRT1 was expressed in chicken embryos of both sexes. DMRT1 gene expressions were up-regulated at four, five and six days of chicken embryos. DMRT1 expression increased at 5-Dpc. of male embryos, however, expression was not signification different in females embryos. Gene expression of FET1, FOXL2, LHX9 and HINT1 were higher in females than in males. The SMC2L1 and SOX9 were expressed in both sexes. Also, to identify the novel sex-determination genes in early chicken subtractive embryos, cDNA libraries from male-minus-female and female-minus-male 3.5 Dpc. embryos cDNA were established. Gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA) and TIGR gene indices (The Institute for Genome Research, USA). A total 548 of colonies in male-minus-female library and 79 sequences were annotated. However, a total of 589 of colonies in female-minus-male library and 16 sequences were annotated. Sequences were homologous to the steroid 5£\-reductase protein (SRD5A1) using BLASTx in male-minus-female subtractive library. The SRD5A1 may play a sex-differentiation role in male chicken. We need more study to know function of steroid 5£\-reductase protein in future.

Drought is a major constraint for attaining economic yield in tree crops. As an initial step to understand molecular response to water-deficit-stress in trees, gene expression in response to water stress was quantified using real-time RT-PCR. The specific objectives established for this to were I. to identify and characterize the genes induced by drought stress in Aleppo pine (Pinus halepensis) and II to identify and quantify the differentially expressed genes in different populations of Loblolly pine (Pinus taeda.L) due to water deficit (chapter III). Results of these studies may be used to identify candidate genes for future breeding programs against water-deficit-stress.

Der Folsäureantagonist Methotrexat (MTX) wird zur Behandlung onkologischer und rheumatoider Erkrankungen eingesetzt. Die Aufnahme des Methotrexats in die Zielzelle ist dabei Vorraussetzung für die Bindung an seine intrazellulären Zielstrukturen und erfolgt über verschiedene Transportsysteme. In diesem Zusammenhang ist bei entsprechenden Plasma-konzentrationen von MTX der Reduced Folate Carrier (RFC1) von besonderer Bedeutung. 1994 konnte erstmals die cDNA dieses Transporters aus Maus- und Hamstergewebe isoliert werden. Die cDNA für einen mit dem RFC1 identischen hepatozellulären MTX-Transporter der Ratte wurde 2000 kloniert. Vorhergehende Gen-Expressionsstudien zeigten, dass die RFC1-mRNA ubiquitär gebildet wird. Die Proteinexpression wurde jedoch bisher nur in ausgewählten Geweben der Maus untersucht. Systematische Arbeiten, in denen in vergleichender Weise sowohl die RFC1 Gen- als auch die Proteinexpression in allen Geweben mit einer möglichen Relevanz für die Folat- und Antifolataufnahme, Speicherung und Eliminierung untersucht werden, fehlten bisher. Insbesondere die Expression des RFC1-Proteins der Ratte (rRFC1) mittels immunologischer Verfahren ist bisher nicht beschrieben worden. Ziel dieser Arbeit war es daher, die Gen- und Proteinexpression des rRFC1 in ausgewählten Geweben der Ratte darzustellen. Dieses schließt die Generierung spezifischer Antiseren gegen den rRFC1 als ersten Schritt mit ein. Es wurden geeignete antigene Aminosäuresequenzen des rRFC1 bestimmt und die entsprechenden cDNA Sequenzen wurden amplifiziert und in einen geeigneten Expressionsvektor kloniert. Rekombinante rRFC1 Fusionsproteine konnten mittels E. coli Zellen hergestellt und anschließend aufgereinigt werden. Nachfolgend wurden entweder die rRFC1 Fusionsproteine oder die rRFC1 spezifischen Peptide, welche von dem Affinitätspeptid separiert worden waren, für die Immunisierung von Kaninchen verwendet Drei Antiseren mit ausreichender Reaktivität und Spezifität konnten gewonnen und mittels Affinitätschromatographie aufgereinigt werden. Die erhaltenen Antiseren sind gegen die intrazellulären N- und C-terminalen Regionen (ID1, ID7) bzw. gegen die erste extrazelluläre Schleife (OD1) gerichtet. In Western-Blot Studien konnte mittels dieser Antiseren für den rRFC1, der in transfizierten Nierenepithelzellen (MDCK-rRFC-HA) stabil exprimiert wurde, ein Molekulargewicht von 71 kD für die glykosylierte Form und von 53 kD für die unglykosylierte Form ermittelt werden. Weiter konnte belegt werden, dass das Protein in MDCK-rRFC1-HA Zellen überwiegend in der glycosylierten Form vorliegt. Mittels RT-PCR Analysen wurde die Genexpression des rRFC1 in allen untersuchten Geweben nachgewiesen. Besonders hohe mRNA-Gehalte waren in Thymus, Niere und Milz vorhanden, während in Herz- und Muskelgewebe sowie in Leukozyten nur ein Signal nahe der Nachweisgrenze detektierbar war. Durch immunhistologische Untersuchungen konnten die rRFC1 Proteinexpression und beträchtliche Unterschiede in der Signalintensität bestätigt werden. Zusätzlich konnten neue Informationen über die unterschiedliche subzelluläre Lokalisation gewonnen werden: so konnte eine starke Expression des Transporters in der apikalen Membran von Dünn- und Dickdarmmukosa dargestellt werden, während die ebenfalls starke Färbung in der Niere auf den Bereich der basolateralen Membran der Tubuli beschränkt war. In der Leber war eine Expression mittlerer Intensität im Bereich der Lebertrias erkennbar. Während in der Milz nur in der roten Pulpa das RFC1-Protein detektiert wurde, konnten im Thymus sowohl in der Rinde als auch im Mark positive Zellen nachgewiesen werden. Im Hoden konnte der Transporter in den Sertoli-Zellen dargestellt werden. Eine starke Expression des Transporters wurde im Gehirn im Bereich der apikalen Membran der Ependymzellen des Plexus choroideus nachgewiesen. In der Skelettmuskulatur und im Herzgewebe beschränkte sich die Expression des rRFC1 auf das Perimysium des Muskelgewebes und auf kleinere Gefäße des Muskel- und Herzgewebes. In dieser Arbeit konnte somit gezeigt werden, dass der RFC1 der Ratte ubiquitär exprimiert wird, wobei die Expressionsstärke jedoch stark variiert. Die beobachtete Gewebslokalisation des RFC1 belegt sowohl dessen zentrale Rolle in der Folathomöostase als auch in der MTX vermittelten Organtoxizität und Pharmakokinetik, insbesondere bei der intestinalen Resorption sowie der hepatischen und renalen Exkretion.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

Charles University, Faculty of pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Ženklová Supervisors: Prof. Fabio Altieri and PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Involvement of PDIA3 in oxidative stress response PDIA3 is a member of the protein disulfide isomerase family (PDI) and it is a stress- responsive protein. It is also involved in various cellular signalling pathways and has various functions in the cell. The best-known location is in the endoplasmic reticulum where it plays a major role mainly in the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class I. However, its existence has also been described in many other cell compartments, such as nucleus, mitochondria, cell surface or cytosol, where it interferes in various processes. While in some instances these roles need to be confirmed by further studies, a lot of observations confirmed its involvement in the signal transduction (for example releated with STAT protein) from the cell surface and the regulatory processes in the nucleus. Recent studies have also confirmed its increased expression in various pathological states. The aim of our work was to find out what is its role in the exposure of the MDA-MB 468...

SOUZA, Isabel Cristina da Cósta. Caracterização bioquímica de calos de espécies laticíferas em resposta ao estresse salino e análise da transcrição de osmotinas. 2015. 107 f. Dissertação (mestrado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2015. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-03-22T17:57:48Z No. of bitstreams: 1 2016_dis_iccsouza.pdf: 2040395 bytes, checksum: f08a9aac46cd6e718bf109abee257b2f (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-04-28T22:22:53Z (GMT) No. of bitstreams: 1 2016_dis_iccsouza.pdf: 2040395 bytes, checksum: f08a9aac46cd6e718bf109abee257b2f (MD5) / Made available in DSpace on 2016-04-28T22:22:53Z (GMT). No. of bitstreams: 1 2016_dis_iccsouza.pdf: 2040395 bytes, checksum: f08a9aac46cd6e718bf109abee257b2f (MD5) Previous issue date: 2015 / Calotropis procera e Cryptostegia grandiflora are laticiferous plants. It was found osmotin protein. The literature shows that the osmotinas are associated to plant defence mechanisms in situations of biotic or abiotic stress. However, there are still several inconsistencies in this hypothesis. In this context, it was used in vitro tissue culture techniques as model to assist in the understanding of how the C. procera and C. grandiflora callus cells respond to salt stress in biochemical terms, and whether the transcripts level for osmotin has raised in response to exposure to NaCl. It was added NaCl to the culture medium of Murashige e Skoog (MS) in increasing concentrations (0, 20, 40, 60 e 80 mM). The results show that callus treated with 80 mM NaCl have reduced the growth and the humidity percentage of respectively 33% and 10% in C. procera and 83% and 39% in C. grandiflora compared to the control treatment callus. Under the same conditions, it was seen an increase in ions concentrations of Na+ and Cl-, 98.9% and 98% in C. procera and 98.8% and 96% in C. grandiflora respectively. It was also seen a reduction in K+ level in callus treated with 80 mM NaCl, 43% in C. procera and 18% in C. Grandiflora, when compared to the control. The callus treated with 80 mM NaCl, showed a tendency of the proline accumulation and soluble sugars, increasing 26% and 37% in C. procera callus and 55.4% and 45% in C. grandiflora callus, respectively, when compared to control conditions. The increase in the activity of enzymes that break H2O2 has been observed in C. grandiflora callus under the salt stress, suggesting a possible oxidative damage, this increase was 73% in the ascorbate peroxidase activity and 62% in guaiacol peroxidase activity when compared to the activity of enzymes of the control callus with the treaty in 80 mM NaCl. None connection was seen between changes in the activity of the enzymes of the oxidative system and the salt stress in C. procera callus. It was evaluated the behaviour of osmotin in the osmotin transcription at 0, 2, 12, 24, 48 hours and at 4, 7, 14, 28 days of callus contact under stress. The osmotin transcripts were observed from 12 hours of contact of callus in 80mM NaCl in both species. However, it was not found osmotin by electrophoresis assays, dot blotting and mass spectrometry in the protein extracts of C. procera and C. grandiflora callus grown in control conditions and in 80 mM NaCl. Thus, the salt stress evaluation using in vitro cell model study was effective, providing cellular behaviour information for these two laticifers plants species showing their physiological, biochemical and molecular changes. The results suggest that the induced salt stress has favoured the increase of osmotin gene expression in both cases and suggests a possible relationship between osmotin of these species with the protection to salinity conditions. The failure to detect the proteins corresponding to genes provides the conception of several new hypotheses to be validated. / Calotropis procera e Cryptostegia grandiflora são plantas laticíferas. Em seus fluidos laticíferos, foram encontradas proteínas do tipo osmotina. A literatura reporta que osmotinas são proteínas relacionadas com mecanismos de defesa vegetal em situações de estresse biótico e/ou abiótico. Entretanto, ainda há várias inconsistências nessa afirmação. Nesse contexto, técnicas in vitro de cultura de tecidos vegetais foram aplicadas como modelo para auxiliar na compreensão de como as células de calos de C. procera e C. grandiflora respondem ao estresse salino, em termos bioquímicos, e se o nível de transcritos para a osmotina seria aumentado em resposta à exposição a NaCl. Para indução desse estresse, NaCl foi adicionado à formulação nutritiva de Murashige e Skoog (MS), em concentrações crescentes (0, 20, 40, 60 e 80 mM). Os resultados mostram que os calos cultivados com NaCl a 80 mM tiveram o crescimento e o teor de umidade reduzidos, respectivamente, em 33% e 10%, em C. procera e de 83% e 39%, em C. grandiflora, em comparação ao seu tratamento controle. Nessas mesmas condições, foi observado um aumento nas concentrações dos íons Na+ e Cl- de, respectivamente, 98,9% e 98%, em C. procera, e de 98,8% e 96%, em C. grandiflora. Foi também observada diminuição no teor de K+ nos calos tratados com NaCl a 80 mM. Essa redução foi de 43%, em C. procera, e de 18% em C. grandiflora, quando comparado ao tratamento controle. Os calos tratados com NaCl a 80 mM, apresentaram uma tendência de acúmulo de prolina e açúcares solúveis, alcançando, respectivamente valores, 26% e 37% maiores em calos de C. procera, e 55,4% e 45% maiores, em calos de C. grandiflora, que aqueles em condições controle. O aumento na atividade das enzimas que degradam H2O2 foi observado em calos de C. grandiflora submetidos a estresse salino, sugerindo um possível dano oxidativo. Esse aumento foi de 73%, para a ascorbato peroxidase, e de 62% para a peroxidase do guaiacol, nos calos tratados com NaCl a 80 mM, em relação ao controle. Não foi observada qualquer alteração significante na atividade das enzimas do sistema antioxidativo em razão do estresse salino em calos de C. procera. Em relação à transcrição da osmotina, foi avaliado o perfil de seus transcritos nos intervalos de tempo de 0, 2, 12, 24, 48 horas e de 4, 7, 14 e 28 dias sob estresse. Os transcritos de osmotina foram observados a partir de 12 horas de contato dos calos com NaCl a 80 mM, em ambas as espécies. Contudo, nos extratos proteicos dos calos de C. procera e C. grandiflora cultivados em condições controle e de 80 mM de NaCl, não foi detectada a presença da proteína osmotina quando avaliado pelos ensaios de eletroforese, Dot blotting e espectrometria de massas. Assim, a avaliação do estresse salino utilizando como modelo de estudo células in vitro foi eficiente, fornecendo informações do comportamento celular de duas espécies laticíferas, mostrando suas alterações fisiológicas, bioquímicas e moleculares. Os resultados sugerem que o estresse salino favoreceu o aumento da transcrição do gene da osmotina em calos das duas espécies em estudo e permite propor uma possível relação das osmotinas dessas espécies com a tolerância à salinidade. A falha em detectar as proteínas correspondentes aos genes propicia a concepção de várias novas hipóteses a serem validadas.

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-29Bitstream added on 2014-06-13T20:33:23Z : No. of bitstreams: 1 silva_ds_me_araca.pdf: 824748 bytes, checksum: a5191c74f89f8870aa31a3483beae50a (MD5) / O tumor venéreo transmissível canino (TVTC) é uma neoplasia transmitida entre cães saudáveis pelo contato direto de pele e/ou mucosas lesionadas. Face aos escassos estudos relacionados aos eventos celulares envolvidos nas fases de crescimento do TVTC, o presente estudo teve por objetivo identificar a presença do gene TP53 e o RNAm referente a proteína codificada, além de detectar a expressão das proteínas p53, Bcl-2 e p63 em cortes histológicos de 13 amostras de TVTC. Com relação à evolução da neoplasia, 46% das amostras foram consideradas em fase de progressão e 54% no estágio de regressão. Foram utilizadas as técnicas de hibridização in situ (ISH) e RT-PCR in situ, que demonstrou a presença do DNA homólogo ao TP53 e seu respectivo RNAm em 92,30% das amostras. A expressão das proteínas p53, p63 e Bcl-2 foram detectadas em 50%, 70% e 100% das amostras, respectivamente. A p63 foi expressa de forma evidente nas amostras em regressão, porém a p53 e a Bcl-2 não apresentaram relação com o estágio evolutivo do tumor e provavelmente não podem ser analisados como fatores de prognóstico do TVTC. Observou-se, nesse estudo que, através das técnicas de ISH e RT-PCR in situ foi possível detectar o DNA do TP53 e seus transcritos, porém esse fato não significou a transcrição da p53, devido aos baixos níveis de expressão nas análises quantitativa e qualitativa nas amostras de TVTC / The canine transmissible venereal tumor (CTVT) is transmitted by direct contact of skin or mucosal presenting lesions. In fact, few reports have been found describing the cellular immune response related to the evolution of the tumor. The objective of this study was to identify the TP53 gen and its transcription in CTVT in different stages of evolution, collected from dogs (N=13) examined at veterinary school, UNESP, Aracatuba, SP, Brasil. In addition, it was also evaluated the expression of p53, p63 and Bcl-2 in histological sections by the use of immunohistochemystry assay. The p53, p63 and Bcl-2 were evident in 50, 70 and 100% of analyzed samples. Regarding to tumor evolution, 6 out of 13 were considered in a progressive stage (46%), was list 7 out of 13 were classified in a regressive stage (54%). The use of in situ hybridization and reverse transcriptase polymerase chain reaction in situ (RT-PCR), revealed that TP53 was present in all samples and P63 was more expressed than p53. Take all results together, the real role of those marker are extremely important to understand the biological behavior and to improve therapeutic procedures for CTVT